Anti ia ie clone m5 114

Cells were stained in 1X PBS containing 2% FBS and 2 mM EDTA for 30 min at 4°C and then fixed with 2% PFA for 15 min at 4°C. Single-cell preparations were stained with antibodies to the following markers: anti-Ly6C (clone HK1.4, BioLegend), anti-IgA (clone mA-6E1, eBioscience), anti-Ly6G (clone 1A8, BD), anti-SiglecF (clone E50-2440, BD), anti-CD11c (clone HL3, BD), anti-CD45 (clone 30-F11, BioLegend), anti-IA/IE (clone M5/114.15.2, BD), anti-B220 (clone RA3-6B2, BioLegend), anti-CD138 (clone 281-2, BioLegend), anti-CD64 (clone X54-5/7.1, BD), anti-CD11b (clone M1-70, BD) and anti-CD5 (clone 53-7.3, BD). Dead cells were excluded from analysis using the viability dye BD Horizon Fixable Viability Stain 510 (BD). Data were acquired in an LSRFortessa X20 cytometer (BD Biosciences) and analyzed using FlowJo v10.0.7 software (BD Biosciences).

Single-cell suspensions obtained from lungs homogenates were first incubated with anti-CD16/32 (clone 93 BioLegend, San Diego, CA, USA) to block non-specific antibody interaction with Fc receptors. Lungs alveolar macrophages were identified using anti-CD45 (clone 30F11; BD Biosciences, San Diego, CA, USA), anti-CD11b (clone M1/70; BD Biosciences), anti-F4/80 (clone BM8; BioLegend, San Diego, CA, USA), anti-Siglec F (E50-2440; BD Biosciences, San Diego, CA, USA) and anti-CD11c (clone HL-3; BD Biosciences, San Diego, CA, USA). A viability dye (LIVE/DEAD Fixable; Molecular Probes) was added to discriminate live cells. Autofluorescent cells were excluded by gating on CD45⁺and FITC^-. Thereafter, total dendritic cells were identified with anti-CD11c, anti-CD11b, anti-IA/IE (clone M5/114.15.2, BD Biosciences), anti-CD45 and anti-F4/80. Flow cytometry was performed using BD LSR II (BD Biosciences, Ontario, Canada) and data analysed with FACSDiva software Version 6.1.2 (BD Biosciences, Ontario, Canada).

Cells were preincubated with a LIVE/DEAD Fixable Near-IR cell stain kit (Invitrogen) and with Mouse BD Fc Block™ (≤1 μg/million cells in 100 μl, BD Biosciences) at 4 °C for 10 min.
The following antibodies were purchased from BD Bioscience: anti-CD45 (clone 30-F11), anti-CD19 (clone 1D3), anti-CD3 (clone 17A2), anti-CD25 (clone 7D4), anti-CD11c (clone HL3), and anti-I-A/I-E (clone M5/114.15.2). The following antibodies were purchased from BioLegend: anti-CD45.1 (clone A20), anti-CD45.2 (clone 104), anti-CD4 (clone GK1.5), anti-CD8 (clone 53-6.7), anti-CD11b (clone M1/70), anti-CD115 (AFS98), anti-CD11c (clone N418), F4/80 (clone BM8), anti-Ly-6G (clone 1A8), and anti-CD69 (clone H1.2F3). The following antibody was purchased from eBioscience: anti-CD4 (clone GK1.5). Corresponding isotype-matched irrelevant specificity controls were purchased from BD, BioLegend, and eBioscience. Reticulocytes were stained with thiazole-orange (Sigma-aldrich). Multiparameter analysis was performed with an LSRFortessa analyzer (BD Biosciences), a SP6800 Spectral Analyzer (Sony) or an Aurora 5 L spectral flow cytometer (Cytek). The data were analyzed using FlowJo software (version 10.7.1) and FCS express 7 (De Novo software).